The broad long term objective of this proposal is to understand the molecular mechanism underlying low level lead (Pb) toxicity and to determine the precise role of Pb2+ in mediating detrimental changes in cellular biochemistry. The primary focus of this application is to determine the interactions of Pb2+ with protein kinase C (PKC). Why Pb2+ and PKC? Pb toxicity is a major pediatric health issue in the United States. Pb is well recognized as an important neurotoxin with exposure, at various levels, leading to mental retardation, cognitive impairments, and maladaptive behavior. PKC is a family of highly conserved signal transduction elements first described as a Ca2+/phospholipid-dependent kinase activity. The PKCs play important roles in differentiation, development, responses of cells to hormones and neurotransmitters, and the processes of learning and memory. Several cellular effects of Pb2+ are mediated through activation of PKC. Pb2+ activates PKC at 10[-11] M, 5000-fold lower than the concentration of Ca2+ required to activate PKC. PKC is perhaps the highest affinity target of Pb2+ identified to date. The Ca2+-dependent isoforms of PKC have a high affinity Ca2+/phospholipid binding domain and all PKC isozymes contain high affinity "Zn2+ finger" regions. The proposed studies are designed to determine if Pb2+ acts at one or more of these sites to activate PKC and the effects of such substitutions on the behavior of this enzyme. These studies are made possible through the use of the high affinity divalent cation chelator 1,2-bis(2-amino-5fluorophenoxy) ethane N,N,N',N'-tetraacetic acid (5F-BAPTA). 5F-BAPTA serves as both a metal buffer and as a metal indicator when observed by 19F NMR. Using 5F-BAPTA, pM-free concentrations of Pb2+ can be set and confirmed alone with nM concentrations of Zn2+ and micromolar Ca2+. The current study is designed to answer the following questions. 1) What isoforms of PKC are activated by Pb2+? 2) How does Pb2+ activate PKC? 3) How does Pb2+ activation alter the biochemical characteristics of PKC? These studies will be performed on highly purified, genetically-defined PKC isozymes from cDNA inserted into baculovirus vectors and expressed in Sf9 cells. This study will resolve the high affinity interactions of heavy metals with the critically important PKC family of signal transduction proteins, describe the possible molecular mechanisms underlying the toxicity of "low levels" of Pb, and provide a model approach to study high affinity heavy metal interactions with proteins and biochemical processes.